The invention relates to the DNAs of papillomaviruses, and more particularly to the probes derived from these papillomaviruses, as well as procedures for their implementation in the in vitro diagnosis of papillomavirus infections.
The expression "papillomavirus" covers a large number of viruses, which have in common the property of being held responsible for several forms of viral infection extending from relatively benign epidermal and mucosal warts to hyperplasias liable to degenerate into intra-epithelial neoplasias and cancers of the skin and mucous membranes. Of these papilloma infections, mention will also be made more especially of epidermodysplasia verruciformis, which will sometimes be designated below by the expression "EV".
Quite a number of types of papillomavirus have already been described. The European patent application No. 85. 402362.9-2105, filed on Nov. 29, 1985, and published on Sep. 10, 1986, as No. 0192001, describes quite a number of new types and subtypes of papillomaviruses which were isolated from warts and macular lesions liable to give rise to the development of skin cancer in a high proportion of those patients affected and from pre-cancerous lesions of the skin, and lesions in oral hyperplasia and in a cancer of the uterine cervix.
The major role of various types of human papillomaviruses (HPV) in the genesis of neoplasia is an accepted fact. Their pathogenicity is also influenced by various genetic factors, and in particular immune and/or extrinsic factors such as actinic radiation.
Several new types and subtypes of papillomaviruses were identified in the previous application. The use in more refined in vitro diagnostic techniques of the DNAs of these new papillomaviruses, used individually or in combination with each other and/or with the DNAs of previously known HPVs has also been described in this application.
The general observation was made in the European application that papillomaviruses, although differing very much among themselves, have sizes of the order of 7,000-8,000 base pairs. Furthermore, their genomes may present certain degrees of homology, assessed as percentages of homology between types and subtypes of papillomaviruses in hybridization assays carried out under so-called non-stringent conditions or also under stringent hybridization conditions.
It has been said that the papillomaviruses which present percentages of homology of less than 50% under stringent conditions belong to different types. Viruses for which percentages of homology higher than 50% are observed under the same stringent conditions are believed to belong to the same type.
The hybridization assays under non-stringent conditions involve placing in mutual contact DNAs derived from two isolates of virus under the conditions described by HEILMAN, C. A. et al. 1980, J. Virol., 36, 395-407, and CROISSANT et al., 1982, C. R. Acad. Sci. Paris, 294, 581-586 (heteroduplex molecules).
Performance of hybridization assays under stringent conditions involves placing in mutual contact DNAs derived from two isolates of virus under the conditions described by HEILMAN, C. A. et al., 1980, and KREMSDORF, D. et al. ((1982), J. Virol. 43, 436-447 and 1983, J. Virol., 48, 340-351) and DAVIS, R. W. et al., 1971, Methods Enzymol., 21, 413-418 heteroduplex molecules).
Based on these considerations, several new viruses were described in the previous application. Similarly, genetic recombinants have been described containing all or part of the genomes of these viruses (designated DNA-HPVs). The invention of the previous application consequently related to each of the DNA-HPVs chosen from among the totality of DNAs which were of a molecular size ranging from 7,000 to 8,000 base pairs and which were characterized by restriction maps which are presented in the figures also reproduced in this application and which related more particularly to the DNA-HPVs obtained from papillomaviruses and which are designated HPV2d, HPV10b, HPV14a, HPV14b, HPV15, HPV17a, HPV17a, HPV17b, HPV19, HPV20, HPV21, HPV22, HPV23, HPV24, HPV28, HPV29, HPV31, HPV32, HPV-IP2 and HPV-IP4.
The invention of the previous application also related to mixtures or "cocktails" of DNA-HPVs isolated from the new papillomaviruses or from ones already known at the time of the filing date of the previous application, or hybridization probes containing them and which may be put to use for the more effective diagnosis of various classes of infections, and even for the diagnosis of the level of risk which accompanies the discovery in a patient of specific papillomaviruses. The number of the probes for papillomaviruses described in the previous application, to which are added those constituted from the genomic DNAs of papillomaviruses which had already been isolated and their combinations in defined mixtures, thus makes it possible to perform more refined diagnoses, in particular, by enabling a clear distinction to be made between the various classes of infection and the various types of papillomavirus to which they may be ascribed, or the infections which are liable to develop as a result of the influence of such papillomaviruses and, within a given class of specific infections, by providing a better diagnosis of the degree of risk of these infections being transformed into more serious diseases. In particular, the invention of the previous application furnished reagents which make it possible, in cases of infections manifesting themselves as cases of epidermodysplasia verruciformis, to assess more precisely the degree of risk that the latter will develop into epidermal cancers.
Therefore, the invention of the previous application related more particularly to diagnostic mixtures; a group of preferred mixtures was defined as containing:
1) at least the DNA of HPV2d, PA0 2) at least one of the DNAs of HPV10b, 28 and 29, PA0 3) at least one of the DNAs of HPV17, 24, PA0 4) at least one of the DNAs of HPV14, 15, 17, 19, 20, 21, 22, 23 and IP4, PA0 5) at least one of the DNAs of HPV15 and 17, PA0 6) the DNA of HPV24, PA0 7) the DNA of HPV14, 32 and IP4, PA0 8) the DNA of HPV31, PA0 9) the DNA of HPV32, PA0 10) at least one of the DNAs of HPV16, 18 and IP2,
it being understood that the DNAs of the ten groups were chosen so that the groups were different from each other under all circumstances.
In group 10), the DNA of hPV-IP2 is preferably associated with the DNA of HPV16 or HPV18.
The restriction maps of the DNA-HPVs concerned are presented in the FIGS. 1 to 10 of the appendices.
The restriction maps give the positions of the cleavage sites of various restriction endonucleases. The origin of a map is generally constituted by a unique cleavage site. The distances from the origin are expressed as percentages of the length of the genome.